The broad purpose of this research program is to determine the fundamental relationships between the structures of active sites in metalloproteins and function. This collaborative supplement application builds on the existing research efforts in Hydrogen Bonding Cavity Motifs about Metal Ions (2 Ro1 GM050781-21) by offering a new approach that combines both synthetic and biological chemistries with molecular biology to create artificial metalloproteins that control the microenvironments (secondary coordination sphere) surrounding metal ions. Protein hosts streptavidin and avidin serve as new binding sites for synthetic metal complexes: these proteins do not normally bind metal ions but have an unusually high affinity for biotin (Ka ~ 1013 M-1). This specific binding will be used to direct biotinylated synthetic metal complexes to specific locations within the proteins. The protein hosts will control the microenvironments around the metal complexes through non-covalent interactions, particularly hydrogen bonds (H-bonds). The strength of this integrated approach is the ability to independently tune the properties of the artificial metal cofactors (through chemical methods) and the hosts (through molecular biology methods) to readily provide information on essential structure-function relationships. The combined chemical and genetic (chemogenetic) approach allows access to a diverse group of artificial metalloproteins that can directly address questions on how active site structures create specific H-bonding networks about metal ions. Our approach allows for the confinements of two distinct metal complexes at fixed (but close) locations within the proteins, as the secondary coordination sphere is systematically modulated. We can thus explore how differences in metal-metal distances and H-bonding networks affect dioxygen binding and activation, including detecting high-energy transients that are often difficult to observe. Long-term goals include developing structure function relationships in metal-assisted oxidative catalysis. Metalloproteins perform functions not yet achieved in other types of systems, including artificial metalloproteins. Our hypothesis is that the lack of control of the secondary coordination sphere is a major obstacle to desired functions. Results from structural biology show that non-covalent interactions within the secondary coordination spheres of metalloproteins are instrumental in regulating function. Therefore the function and dysfunction of health-related metalloproteins can be understood in the context of changes in their microenvironments. It is still unclear, even in biomolecules, how non-covalent interactions are able to influence metal-mediated processes. Investigations into these effects require basic structural, functional, and mechanistic studies in which the effects of individual components can be analyzed separately. We propose a new chemogenetic approach whereby site-specific modulations in structure of the protein host and synthetic metal complex can be readily accomplished, in order to establish correlations with function these types of studies will lead to fundamental insights into biochemical processes. Ultimately, this research will provide insights into the properties of biological catalysts and lead to new classes of artificial catalysts that incorporate the exquisite control of reactivity characteristic of metalloenzymes.